Method and apparatus for monitoring the state of health of dairy cows

ABSTRACT

The present invention relates to methods and apparatuses for monitoring the state of health of dairy cows, in particular of entire dairy herds. The method is based on analysing the haptoglobin (HP) biomarker and part of the polymeric immunoglobulin receptor (PIGR), the secretory component (Secretory Component, SC), in a milk sample. In particular, the claimed method and apparatus of the invention make it possible to diagnose mastitis or systemic diseases which occur outside the udder on the basis of the protein biomarker described here. The invention therefore makes it possible to regularly monitor the general state of health of a dairy herd. The present invention relates to non-invasive diagnostic methods and to apparatuses and diagnostic kits for carrying out these methods.

The present invention relates to methods and apparatuses for monitoring the state of health of dairy cows, in particular of entire dairy herds. The method is based on analysing the haptoglobin (HP) biomarker and part of the polymeric immunoglobulin receptor (PIGR), the secretory component (Secretory Component, SC), in a milk sample. In particular, the claimed method and apparatus of the invention make it possible to diagnose mastitis or systemic diseases which occur outside the udder on the basis of the protein biomarker described here. The invention therefore makes it possible to regularly monitor the general state of health of a dairy herd. The present invention relates to non-invasive diagnostic methods and to apparatuses and diagnostic kits for carrying out these methods.

DESCRIPTION

The latest technical methods are making it possible to cost-efficiently cultivate increasingly large dairy herds. Moreover, the use of automated milking systems can drastically reduce staffing requirements. However, this means that daily health checks can only be performed on the cows to a limited extent or not at all. Automated health monitoring is one possible solution to this problem. This can be done through the detection of certain health markers in the milk. Suitable markers include acute-phase proteins, such as HP, since their concentrations rise very quickly in the early phase of an immune response. HP measurements in milk are not currently done routinely in agriculture or in veterinary laboratory diagnostics.

Health monitoring after calving takes the form of a clinical examination 7-10 days after calving as well as a puerperal checkup (performed 20 to 28 days after calving) by the herd manager or trained barn staff. This involves assessing the general health, body temperature, lochia, milk production, as well as milk and ketones in urine, if applicable. Udder health is assessed daily by the milking staff at the milking stand. During the monthly milk production assessment, parameters such as cell count, uric acid content, and fat and protein content of the milk are collected in order to assess udder health and the metabolic condition of the individual animal and of the herd. A growing number of farms are able to determine the cell count from the automated milking system at each milking. Although there are approaches for routine detection of health parameters using various methods, they are only able to indicate the metabolic condition, a specific disease or the udder health of the animal.

Farm health monitoring is therefore highly subjective and labor-intensive. Existing solutions rely on the collection and evaluation of parameter combinations (milk conductivity, milk production, movement pattern, resting times, progesterone concentration, ketone bodies, lactate dehydrogenase, fat, protein, lactose and uric acid in milk) using the corresponding measurement techniques and multifunctional herd management programs, such as FullExpert (Lemmer-Fullwood). This enables identification of conspicuous animals (estrus, lameness, miscarriage, abomasal displacement, ketosis, mastitis). However, these systems are labor-intensive and expensive to procure. A veterinarian is consulted in the event of problematic results. The measurement of clinically relevant chemical, metabolic and endocrinological parameters in animal blood is done during routine checkups in veterinary laboratory diagnostics, but can only be performed by a veterinarian on selected, conspicuous or already diseased animals. Parameters used routinely in clinical chemistry can only provide an overall indication of a cow's condition in combination.

Approaches already exist for evaluating the health condition of a dairy cow more easily, quickly and objectively. This is done by measuring acute-phase proteins in the blood or milk. HP is the most frequently examined acute-phase protein in cattle. In the presence of mastitis, the HP concentration is significantly increased in milk as well. However, to date HP in milk has only been discussed as a potential indicator of mastitis.

SC is not an acute-phase protein, but rather forms part of a transmembrane receptor for polymeric immunoglobulins, PIGR, in secretory mucosal epithelial cells and also in the udder. During the binding of polymeric immunoglobulin (Ig)A or IgM, the antibody receptor complex is channeled from the lateral to the apical side of the epithelium via transcytosis. There, the receptor is cleaved enzymatically to release SC and IgA or IgM. This is how IgA is transported into the milk. During peripartum immunosuppression and early lactation, dairy cows are especially susceptible to infectious diseases that do not affect the udder (systemic diseases) such as uterine, hoof, or respiratory infections. Abomasal displacement is also not uncommon. Diagnostic examinations for these diseases are routinely done with animal blood, which requires veterinary assessment and treatment. Since a milk sample can be obtained with significantly less effort, it is in a farmer's economic interest to be able to detect systemic diseases based on this sample medium.

Health management teams at growing dairy farms are always looking for alternative solutions for herd monitoring. In practice, there are numerous parameters that enable the identification of conspicuous cows (estrus, lameness, miscarriage, abomasal displacement, ketosis, mastitis) based on daily, automated measurement at the farm. To date there is no validated milk biomarker to analyze the general state of health. Performing analysis on milk significantly simplifies the sampling process, which in turn makes it possible to perform the measurement at the farm. In particular, it should be emphasized that in contrast to taking a blood sample, the present solution does not require a veterinarian, which positively affects costs and is less of a hindrance to the overall agricultural production process. Accordingly, the problem that the present invention seeks to solve is to provide new approaches for the health management of dairy herds in which it is possible to perform simplified routine checks of large dairy herds that can be done without a veterinarian.

In a first aspect, the identified problem is solved by a non-invasive method for monitoring the state of health of a dairy cow, comprising the steps:

-   -   (a) Providing a milk sample from the dairy cow,     -   (b) Measuring the concentration of one or more biomarkers         selected from among HP and PIGR (preferably SC) in the milk         sample,     -   (c) Comparing the measured concentration from (b) with a         reference value of the one or more measured biomarkers, wherein         a deviation from the reference value indicates an unhealthy         condition of the dairy cow.

In the context of the present invention, the determination of the PIGR marker in a milk sample preferably comprises the determination of the SC of the PIGR. It is therefore preferred that the measurement of the concentration of the biomarker PIGR in step (b) encompass measuring the concentration of the secretory component (SC) in the PIGR.

Preferably the non-invasive method is performed completely ex vivo or in vitro. In this regard, it should be emphasized that the biomarkers of the present invention are analyzed in a milk sample, meaning that the method can be performed without invasive sampling and therefore, without a veterinarian. This allows for expanding the present method to large herds of dairy cattle and to regular (monthly) tests, which is not cost-effective with, for example, analysis of biomarkers in a blood sample.

The terms “protein biomarker,” “biomarker” and “marker” are used synonymously for the purposes of the present description. The terms preferably refer to the concentration of individual, or combinations of, biological molecules such as proteins, nucleic acids, carbohydrates, etc. In particular, the present disclosure pertains to proteins as biomarkers. Insofar as the disclosure relates to measuring biomarker concentrations, this is intended to include both a direct measurement of the concentration (number of protein molecules/volume or weight) as well as indirect measurement. In this way, degradation products of the protein markers according to the invention can also be measured, or alternatively, the biomarker concentrations can be inferred based on their biochemical characteristics. Enzymes can be determined through detection of their enzymatic activity, for example.

Insofar as is necessary, the method of the present invention can optionally include in step (b) the measurement of one or more additional biomarkers. The one or more additional biomarker(s) is preferably selected from the group comprising S100 calcium binding protein A9 (S100A9), interleukin (IL-) 18, tumor necrosis factor (TNF-) alpha, lactoferrin (LTF), and Vascular Endothelial Growth Factor (VEGF).

Especially preferred is a method wherein step (b) encompasses the measurement of a combination of two or more biomarkers and the combination of two or more biomarkers is selected from the combinations (i) HP and VEGF, (ii) HP and PIGR (preferably SC), (iii) HP and LTF, (iv) VEGF and PIGR (preferably SC), (v) LTF and PIGR (preferably SC), and (vi) LTF and VEGF. The combination of the markers HP and PIGR (preferably SC) has been shown to be especially advantageous and therefore represents a preferred embodiment of the present invention.

An additional optional and preferred further development of the present invention constitutes a method wherein step (b) encompasses the measurement of a combination of three or more biomarkers, namely HP, PIGR (preferably SC) and a third biomarker selected from the group comprising S100A9, IL-18, TNF-alpha, LTF, and VEGF. The specificity and sensitivity of the method can be further improved through measuring additional biomarkers.

A method described herein is preferred, wherein a deviation of the measured concentration of the biomarker from the reference value indicates mastitis or a systemic disease in the dairy cow, preferably a systemic disease that does not or not exclusively appear on the udder, such as for example minor systemic disease, minor systemic disease with abomasal displacement, serious systemic disease or combinations of these diseases. Preferably the measured deviation is an increased concentration of the biomarker in the sample of a sick cow compared to a healthy cow.

In one aspect, a systemic disease, preferably outside the udder, can be diagnosed based on the disclosed biomarkers [by using] the present invention. Alternatively, however, the invention also relates to the diagnosis of mastitis based on the disclosed biomarkers. For this aspect, there is a preferred embodiment of the invention in which mastitis in a dairy cow is diagnosed by determining a combination of the biomarkers HP and PIGR (preferably SC) or only based on the marker PIGR (preferably SC).

The term “reference value” is intended to be broadly interpreted here and to encompass a plurality of possible comparative values. A suitable reference value is selected based on the diagnostic objective. To identify sick animals, the reference value can be a value for the biomarker in a healthy cow. If the method is used to monitor the progression of a disease or to monitor a course of treatment for a sick cow, the reference value can also be a concentration of the biomarker in the milk of the monitored cow from an earlier point in time—especially before the treatment began. It is especially preferred, however, that the reference value be a threshold value (cut-off) wherein if the measured concentration of the biomarker exceeds the threshold value, it is determined that the cow is not in good health. Depending on how the cut-off is chosen, a non-healthy state can be determined based on a test value that is higher, or equal to and higher than, the cut-off. Additionally, a threshold value specific to the herd can be can be determined wherein a healthy dairy herd to be monitored is tested regularly for the biomarker and based on these values, a “healthy” reference value specific to the herd is determined. If a cow becomes conspicuous due to a higher concentration of the biomarker in the course of regular monitoring, it can be presumed that the cow is not in good health.

In preferred embodiments of the present invention, the threshold values (cut-offs) to distinguish healthy from sick animals can be chosen such that the corresponding biomarker has a specificity of 90% or higher, preferably 92%, more preferably 94% or higher, with a sensitivity of 50% or higher, preferably 60%, 70% or 80% or higher. For example, the threshold value for the marker HP can therefore be approximately 0.4 pg/ml, preferably approximately 0.5 μg/ml and most preferably approximately 0.58 pg/ml. For example, the threshold value for the marker PIGR (preferably SC) can be approximately 5 μg/ml, preferably approximately 8 μg/ml and most preferably approximately 8.2 μg/ml. The threshold value for the marker LTF can be, for example, approximately 80 μg/ml, preferably approximately 100 μg/ml and most preferably approximately 120 μg/ml. The value for the marker VEGF can be approximately 7 μg/ml, preferably approximately 9 μg/ml and most preferably approximately 9.5 μg/ml, for example. The term “approximately” in connection with numerical information preferably refers to a deviation of +/−20% of the specified value, more preferably a deviation of +/−15%, +/−10%, and most preferably +/−5%.

The biomarkers and biomarker combinations described herein have been found to be especially advantageous for diagnosing systemic diseases. In some embodiments, the method is therefore not used to diagnose mastitis, in particular, but rather only to detect systemic diseases. In this regard, with some embodiments, the diseases can be detected at an early stage.

As mentioned above, the non-invasive method according to the invention is suitable for monitoring a treatment of a dairy cow, wherein a reduction in the biomarker concentration during or following a treatment indicates a successful treatment. Since the increase of the biomarkers in the milk described here correlates to a deterioration of a dairy cow's state of health, the method can also be used to monitor the success of treatments for individual sick animals. In this regard, the reference value with which a measured concentration of the biomarker(s) according to the invention in the milk sample is compared, is [equivalent to], for example, a concentration of the biomarker(s) in the milk of the same cow at an earlier point in time, especially at the beginning of the treatment.

It is especially preferred for the present invention that in step (b) both HP as well as PIGR (preferably SC) be measured.

In some embodiments it is preferred that the concentration of the biomarker HP be determined in an undiluted milk sample. In other embodiments the milk sample is a milk sample to which preservatives have been added (e.g., during the milk production test).

The method of the present invention can be used especially on a routine basis for monitoring a dairy cow or a dairy herd. It is therefore preferred that the non-invasive method be performed regularly, preferably monthly, more preferably weekly, and even more preferably several times per week, up to daily.

To carry out the method according to the invention, the milk sample from the dairy cow that is to be analyzed is preferably taken during a milking (non-invasively). With fully automated milking systems, the milk sample from a dairy cow can be (automatically) diverted directly. The milk sample obtained in this manner is then used for the method according to the invention described here.

To measure the concentration of the biomarkers, the present invention can refer back to various technical methods with which one skilled in the art is familiar. In particular, the present disclosure should not be regarded as limited to individual analysis methods. The determination of the concentration of biomarkers in a milk sample can encompass measuring the concentration biochemically by means of a method selected from among Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Fast Protein Liquid Chromatography (FPLC), and High Performance Liquid Chromatography (HPLC), or measuring the concentration immunologically by means of a method selected from among Enzyme-linked Immunosorbent Assay (ELISA), Enzyme immunoassay (EIA), Fluorescence immunoassay (FIA), Chemiluminescence immunoassay (CIA), Radio immunoassay (RIA), Western blot, and peptide arrays, or encompass measuring the concentration spectrometrically by means of a method selected from among Surface Plasma Resonance (SPR), Matrix-assisted Laser Desorption/lonization (MALDI) or Electrospray Ionization (ESI). Immunological methods, especially by means of monoclonal (preferred) or polyclonal antibodies, such as in an ELISA, are especially preferred.

The described method is intended to be used, in particular, to monitor the state of health of one or more dairy cows. It is therefore intended in several embodiments that the method be used to determine an unhealthy condition in a dairy cow. In this embodiment, it is not absolutely necessary for a specific diagnosis to be made by the present invention of the dairy cow categorized as unhealthy. Rather, the disease from which the identified unhealthy dairy cow is suffering can be determined by performing additional subsequent diagnostic procedures. In this embodiment, the method of the present disclosure focuses on detecting conspicuous animals as early as possible through regular assessments according to the present invention. When an unhealthy condition is determined to exist, a veterinarian can be subsequently consulted to establish a specific diagnosis.

In several embodiments, the present disclosure concerns a non-invasive method to monitor the state of health of a dairy herd at a dairy farm, comprising regular performance of a method to monitor the state of health of one or more, preferably all, dairy cows in the dairy cattle herd, according to the non-invasive method for monitoring the state of health of a dairy cow described here.

A dairy cattle herd is understood to mean a group of dairy cattle within a dairy farm of preferably two or more animals, more preferably 5 or more animals, 10 animals, 15 animals, 20 animals, 50 animals or more. The present invention is especially helpful for monitoring dairy cattle herds with more than 10 animals.

The present problem is additionally solved in another aspect by a non-invasive system and/or apparatus for monitoring the state of health of a dairy cow, comprising:

-   -   (a) Means to take a milk sample from the dairy cow;     -   (b) Means to measure the concentration of one or more biomarkers         selected from among HP and/or PIGR (preferably SC) in the milk         sample;     -   (c) Means to compare the measured concentration from (b) with a         reference value of the measured biomarker, wherein a deviation         of the measured concentration from the reference value indicates         an unhealthy condition in the dairy cow.

In several embodiments, the non-invasive system and/or apparatus can comprise additional means to store data and/or means to optically display data, such as a screen.

In several embodiments, the non-invasive system and/or apparatus comprises means for information output. When an unhealthy condition is detected in a cow, the means for information output are suitable to communicate this message. For example, upon detection of an unhealthy condition in a dairy cow, a visual or acoustic alarm can be triggered. Preferably, one or more messages about the identity of the identified unhealthy dairy cow are provided.

Preferably the system and/or apparatus described here is connected to a milking system and comprises means that transfer a milk sample taken from the dairy cow from the milking system to the means provided for receiving a milk sample. One skilled in the art is familiar with automated milking systems that enable milk samples to be taken.

The non-invasive system and/or apparatus according to the present invention preferably comprises additional means to measure one or more additional biomarkers selected from the group comprising S100A9, IL-18, TNF-alpha, LTF, and VEGF. More preferably the system and/or apparatus comprises means to measure the concentration of the biomarker(s) HP and/or PIGR (preferably SC) in the milk sample.

In several embodiments, the means to measure the concentration of a biomarker are selected from among means to perform one of these methods: SDS-PAGE, FPLC, and HPLC, or EIA, FIA, CIA, RIA, Western Blot, and peptide arrays, or SPR, MALDI or ESI. In particular, it is preferred that the non-invasive system and/or apparatus comprise antibodies to measure the concentration of the specified biomarkers.

The non-invasive system and/or apparatus according to the present invention is therefore suitable for performing one of the methods described here.

The problem that the invention seeks to solve is additionally solved by a diagnostic kit for monitoring the state of health of a dairy cow, comprising means to determine the concentration of one or more biomarkers selected from HP and/or PIGR (preferably SC) in a milk sample.

In several embodiments, the diagnostic kit according to the invention comprises means to perform a method selected from among SDS-PAGE, FPLC, and HPLC, or EIA, FIA, CIA, RIA, Western blot, and peptide arrays, or SPR, MALDI or ESI. In several embodiments, the kit comprises antibodies for detection or measurement of the concentration of one of the specified biomarkers. In particular, the diagnostic kit is suitable for performing one of the methods described here to monitor the state of health of dairy cattle.

Below, the present invention is further described on the basis of non-restricting examples.

THE FIGURES SHOW

FIG. 1: mRNA expression of selected markers in milk cells (MZ) and leucocytes (BL) from cows in various states of disease. The concentration of the markers was determined with qPCR and is given as a percentage of the expression of the reference gene cyclophilin B (PPIB) and ubiquitously expressed transcript (UXT). system.: systemic; Erkrank.: disease; MZ: milk cells; BL: leucocytes; * 0.05>p>0.01, and ** p≤0.01.

FIG. 2: Concentrations of potential protein biomarkers in milk. The concentrations were determined using commercially available ELISA kits. n.d.=non-detectable; system.: systemic; Erkrank.: disease; * 0.05>p>0.01, and ** p≤0.01.

FIG. 3: Concentration correlations for the biomarkers HP and LTF in milk and plasma. The concentrations were determined using commercially available ELISA kits. Positive correlations are indicated by the regression lines.

FIG. 4A: ROC curves from selected milk biomarkers. A: ROC analysis of the individual markers in various states of disease.

FIG. 4B: Summarized ROC analysis of all sick animals. system.: systemic; Erkrank.: disease

EXAMPLES

Material and Methods:

Quantification of Protein Biomarkers in Milk and Plasma

Selected proteins in milk and plasma were quantified using commercially available ELISA kits. All HP measurements were done based on undiluted samples since this is sufficient to detect fluctuations of the HP marker at various stages of disease. Precoated plates were incubated with 100 μl of sample (30 min, room temperature (RT)). Purified HP (LeeBioSolutions, St. Louis, Mo., USA) was used as the standard in a range from 8 to 0.125 μg/ml. The plate was washed 3 times in assay wash buffer, then incubated with 100 μl of 1:40 diluted peroxidase-conjugated anti-HP antibodies (30 min, RT). After 3 washings, 100 μL of ready-made tetramethylbenzidine substrate solution (Moss Inc., Pasadena, Md., USA) was added, and incubated for 10 to 30 minutes at RT. The reaction was stopped with 50 μl 9.9% H₃PO₄.

PIGR (SC) was quantified with an ELISA kit to detect bovine PIGR (Life Science USCN Inc.) according to the manufacturer's information. In each case, milk was diluted at a ratio of 1:300 to 1:1,000 for the control samples and 1:5,000 to 1:10,0000 for samples from sick cows. Plasma samples were diluted 1:100,000.

Statistical Analysis

Analysis of the differences between the groups was performed by means of Spearman rank correlations, Receiver Operating Characteristic (ROC) analysis and visualization of the results using SigmaPlot11 Software (Systat Software, Erkrath, Germany). To avoid undesired statistical tendencies, animal samples were randomly selected for analysis with quantitative real-time RT-PCR (qPCR) or ELISA. Data sets were analyzed for standard distribution. If the Shapiro-Wilk test returned a positive result, a t test was performed. The Mann-Whitney Rank Sum test was performed for data without standard distribution. All sick groups were compared to the control group. The data for various diseases outside the udder were combined if a small number of samples had been tested. P values are defined as follows: * 0.05>p>0.01, and ** p≤0.01.

Selection and Evaluation of Potential Biomarkers

The ROC analysis was used to evaluate the discriminatory ability of the biomarkers. An area under the curve (AUC) >0.9 was regarded as highly discriminating and an AUC value <0.6 as non-discriminating. Biomarkers were selected based on the best distinction between minor systemic disease and the control group. Statistical evaluation of biomarkers and marker combinations was performed using TANAGRA open source data mining software. To avoid potential overfitting, cross-validation (CV) was performed (10-fold, 1 repetition). The values for sensitivity, specificity and resubstitution error rate were taken over from the CV. The various diseases were collected into one group. The biomarkers or their combinations were evaluated on the basis of their ability to discriminate sick cows.

EXAMPLE 1 Differential Gene Expression of Biomarkers in Milk

The mRNA expression of individual biomarkers in milk cells was analyzed with qPCR. To confirm the systemic significance of potential biomarkers from the local environment of the mammary gland, the expression pattern of the biomarkers in peripheral leucocytes was examined. Data from groups with minor and serious systemic diseases was combined and tested in the case of a small number of samples in a systemic disease group. FIG. 1 shows the results for the most relevant biomarkers.

EXAMPLE 2 Quantification and Selection of Biomarkers

Based on the results of the previous experiments (microarray, qPCR, etc.), potential biomarkers were selected and quantified at the protein level using commercial ELISA kits. Elevated concentrations of IL-18, LTF, PIGR (SC), TNF-alpha and VEGF were detected in milk in the presence of abomasal displacement, serious systemic disease, mastitis and combinations of the diseases. HP and S100A9, however, showed increased values in the presence of minor systemic disease (FIG. 2). The expression patterns of HP, IL-18 and LTF were also confirmed in plasma in order to determine the validity of the markers for systemic diseases. The correlations of milk and plasma HP and LTF concentrations are shown in FIG. 3. The positive Spearman correlation coefficients (Spearman p) show the relationship between milk and plasma protein concentrations. In addition, the correlation of the strongest biomarkers in the milk was examined. All proteins showed positive correlation of concentrations in the milk in the presence of diseases (Table 1). The best markers underwent further statistical evaluation.

TABLE 1 Correlations of Protein biomarkers in milk and plasma Spearman Correlation of correlation coefficient p n Correlation in milk Milk HP and milk PIGR (SC) 0.67 0.001 71 Milk LTF and milk PIGR (SC) 0.61 0.001 79 Milk HP and milk LTF 0.59 0.001 142 Milk HP and milk VEGF 0.58 0.001 120 Milk LTF and milk VEGF 0.54 0.001 132 Milk VEGF and milk PIGR (SC) 0.41 0.001 79 Correlation in milk and plasma Milk HP and plasma HP 0.78 0.001 121 Milk IL-18 and plasma IL-18 0.38 0.088 21 Milk LTF and plasma LTF 0.33 0.005 69 Correlation in Plasma Plasma HP and plasma LTF 0.59 0.001 63

EXAMPLE 3 Statistical Evaluation of the Biomarkers

The heavily regulated and highly concentrated milk biomarkers HP, PIGR (SC), LTF and VEGF were selected for statistical evaluation. A subgroup of samples in which all four markers had been determined was used for a direct comparison of the results.

Each biomarker alone and combinations of two biomarkers were evaluated. In this regard, 17 control samples and 49 samples from sick cows were used. The discriminatory ability for each disease group was determined by ROC analysis (FIG. 4A, Table 2). HP and PIGR (SC) showed the best distinction of minor systemic disease with an AUC of 0.69 and 0.68. All proteins were highly discriminating for serious systemic diseases and mastitis (AUC>0.9).

TABLE 2 Discriminatory ability of milk biomarkers for various diseases. The data was generated by means of ROC analysis. (Control: n = 17, minor systemic (system.) disease (Erkrank.): n = 17, Abomasal displacement (LMV) (+metabolic disorder): n = 8, serious systemic disease: n = 5, serious systemic disease + abomasal displacement: n = 8, mastitis: n = 11) 95% Confidence Control vs. Sick group AUC interval p HP Minor systemic disease 0.69 0.48-0.89 0.065 LMV (+metabolic disorder) 0.96 0.89-1.03 <0.001 Serious systemic disease 0.99 0.95-1.03 0.001 Serious systemic disease + LMV 0.99 0.95-1.02 <0.001 Mastitis 1.00 1.00-1.00 <0.001 PIGR (SC) Minor systemic disease 0.68 0.49-0.87 0.071 LMV (+metabolic disorder) 0.84 0.64-1.04 <0.05 Serious systemic disease 0.95 0.87-1.04 <0.05 Serious systemic disease + LMV 0.80 0.61-0.99 <0.05 Mastitis 0.99 0.98-1.01 <0.001 LTF Minor systemic disease 0.67 0.48-0.86 0.088 LMV (+metabolic disorder) 0.82 0.62-1.03 <0.05 Serious systemic disease 0.95 0.86-1.05 <0.05 Serious systemic disease + LMV 0.93 0.84-1.03 <0.001 Mastitis 0.98 0.95-1.02 <0.001 VEGF Minor systemic disease 0.57 0.38-0.77 0.459 LMV (+metabolic disorder) 0.99 0.96-1.02 <0.001 Serious systemic disease 0.84 0.58-1.08 <0.05 Serious systemic disease + LMV 0.96 0.90-1.03 <0.001 Mastitis 0.97 0.91-1.03 <0.001

To discriminate between sick and control animals, marker combinations were evaluated using two statistical classification methods, namely multinomial logistic regression (MLR) and k-nearest neighbor classification (K-NN) (Table 4). A second statistical model was applied to avoid potential distortions of the results. HP is the best choice for use as a single biomarker. In combination with PIGR (SC) or LTF, a minor increase in sensitivity or specificity can be achieved. These combinations showed the best results for detecting sick animals.

Practical application of biomarkers requires that the tests have high specificity in order not to overestimate the occurrence of diseases in large dairy cattle herds. An ROC analysis was therefore combined for all sick groups vs. control in order to evaluate the sensitivity (“correct positive”), specificity (“correct negative”), 1-sensitivity (“false negative”) and 1-specificity (“false positive”) of the biomarker determination in milk using various threshold value (cut-off) concentrations. Table 3 shows the values for possible cut-off concentrations with a high specificity of 94%. The corresponding ROC curves are shown in FIG. 4B. At a specificity of 94%, 6% of actually healthy animals would be identified as sick. In the case of determination of HP, PIGR (SC), LTF and VEGF, 18%, 41%, 45% and 33%, respectively, of sick animals would be classified as healthy.

On the basis of this analysis, it could therefore be demonstrated that the determination of HP is suitable for detecting diseases in dairy cattle. A combined measurement with PIGR (SC) or LTF is also possible in order to increase the sensitivity or specificity.

TABLE 3 Disciminatory ability of milk biomarkers for sick animals. The data was generated through ROC analysis. (Control: n = 17, sick: n = 49) 95% Confidence- Cut-Off at 94% Sensiivity at 94% AUC interval P specificity specificity % HP 0.88 0.80-0.96 <0.001 0.58 μg/ml 82 PIGR (SC) 0.82 0.72-0.93 <0.001 8.20 μg/ml 59 LTF 0.84 0.74-0.94 <0.001 120.7 μg/ml 55 VEGF 0.82 0.72-0.92 <0.001 9.50 ng/ml 67

TABLE 4 Evaluation of milk biomarkers and their combinations. The classification was performed by using MLR and K-NN: Control (n = 17) vs. sick (n = 49). Sensitivity, specificity and resubstitution error rates were taken over from the CV (10-fold, 1 repetition). Multinomial logistic regression k-nearest neighbor classification (cross-validation)/% (cross-validation)/% Marker (Combination) Sensitivity Specificity Error rate Sensitivity Specificity Error rate Single marker HP 86 88 13 91 69 15 LTF 84 44 27 82 63 23 VEGF 84 38 28 73 31 38 PIGR (SC) 86 25 30 77 19 38 Marker combinations HP & VEGF 86 88 13 80 94 17 HP & PIGR (SC) 89 81 13 84 75 18 HP & LTF 89 69 17 86 81 15 VEGF & PIGR (SC) 86 63 20 82 56 25 LTF & PIGR (SC) 84 56 23 86 31 28 LTF& VEGF 82 56 25 84 44 27

LIST OF ABBREVIATIONS

AUC Area Under the Curve

BL Leucocytes

CIA Chemiluminescent immunoassay

CV Cross-validation

EIA Enzyme immunoassay

ELISA Enzyme-linked Immunosorbent Assay

Erkrank. Disease

ESI Electrospray Ionization

FIA Fluorescence immunoassay

FPLC Fast Protein Liquid Chromatography

HP Haptoglobin

HPLC High Performance Liquid Chromatography

Ig Immunoglobulin

IL Interleukin

K-NN k-nearest neighbor classification

LMV Abomasal displacement

LTF Lactoferrin

MALDI Matrix-assisted Laser Desorption/Ionization

MLR Multinomial logistic regression

mRNA Messenger ribonucleic acid

MZ Milk cells

PIGR Polymeric immunoglobulin receptor

PPIB Cyclophilin B (reference gene)

RIA Radio immunoassay

ROC Receiver Operating Characteristic

S100A9 S100 calcium-binding protein A9

SC Secretory Component, secretory component of the PIGR

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SPR Surface plasmon resonance

system. Systemic

TNF-alpha Tumor necrosis factor alpha

UXT Ubiquitously-Expressed Transcript (reference gene)

VEGF Vascular Endothelial Growth Factor 

1-4. (canceled)
 15. A non-invasive system for monitoring the state of health of a dairy cow, comprising: a means to measure concentrations of at least two biomarkers including one of the following biomarker combinations (i) to (vi): (i) haptoglobin and Vascular Endothelial Growth Factor; (ii) haptoglobin and Lactoferrin; (iii) Vascular Endothelial Growth Factor and polymeric immunoglobulin receptor; (iv) Lactoferrin and polymeric immunoglobulin receptor; (v) Lactoferrin and Vascular Endothelial Growth Factor; (vi) haptoglobin and polymeric immunoglobulin receptor; in a milk sample, and a processor configured to compare the measured concentrations of the at least two biomarkers with reference values for the at least two biomarkers, wherein a deviation of the measured concentration from the reference values indicates an unhealthy condition in the dairy cow; and an automated or semi-automated milking system configured to obtain the milk sample from the dairy cow during a milking process.
 16. The non-invasive system according to claim 15, further comprising at least one of: a memory and a display.
 17. The non-invasive system according to claim 15, wherein the means to measure concentrations of the at least two biomarkers is selected from the group consisting of chemiluminescent immunoassay apparatus, Fast Protein Liquid Chromatography apparatus, Enzyme immunoassay apparatus, Enzyme-linked Immunosorbent Assay apparatus, Electrospray Ionization Mass Spectrometer, Fluorescence immunoassay apparatus, High Performance Liquid Chromatography apparatus, Matrix-assisted Laser Desorption/Ionization Mass Spectrometer, Radio immunoassay apparatus, Sodium dodecyl sulfate polyacrylamide gel electrophoresis apparatus, Surface plasmon resonance apparatus.
 18. The non-invasive system of claim 15, wherein the means to measure the concentrations of the at least two biomarkers comprises one of the following (i) to (vi): (i) an antibody specific to haptoglobin and an antibody specific to vascular endothelial growth factor; (ii) an antibody specific to haptoglobin and an antibody specific to Lactoferrin; (iii) an antibody specific to Vascular Endothelial Growth Factor and an antibody specific to polymeric immunoglobulin receptor; (iv) an antibody specific to Lactoferrin and an antibody specific to polymeric immunoglobulin receptor; (v) an antibody specific to Lactoferrin and an antibody specific to Vascular Endothelial Growth Factor; (vi) an antibody specific to haptoglobin and an antibody specific to polymeric immunoglobulin receptor.
 19. A diagnostic kit to monitor the state of health of a dairy cow, comprising a means to measure concentrations of at least two biomarkers including one of the following biomarker combinations (i) to (vi): (i) haptoglobin and Vascular Endothelial Growth Factor; (ii) haptoglobin and Lactoferrin; (iii) Vascular Endothelial Growth Factor and polymeric immunoglobulin receptor; (iv) Lactoferrin and polymeric immunoglobulin receptor; (v) Lactoferrin and Vascular Endothelial Growth Factor; (vi) haptoglobin and polymeric immunoglobulin receptor;
 20. The diagnostic kit of claim 19, wherein the means to measure the concentrations of the at least two biomarkers comprises one of the following (i) to (vi): (i) an antibody specific to haptoglobin and an antibody specific to vascular endothelial growth factor; (ii) an antibody specific to haptoglobin and an antibody specific to Lactoferrin; (iii) an antibody specific to Vascular Endothelial Growth Factor and an antibody specific to polymeric immunoglobulin receptor; (iv) an antibody specific to Lactoferrin and an antibody specific to polymeric immunoglobulin receptor; (v) an antibody specific to Lactoferrin and an antibody specific to Vascular Endothelial Growth Factor; (vi) an antibody specific to haptoglobin and an antibody specific to polymeric immunoglobulin receptor. 